1. Technical Field
This invention relates generally to the field of sensors for use in cell and molecule based assays. In particular, the invention provides sensor devices which detect a change in measured impedance between and among electrodes, from which the presence, behavior, quantity or change in cells or molecules in a sample solution can be identified. The sensor devices can be used for monitoring cell or particle attachment, growth and migration and in identifying modulators of cell attachment, growth and migration. The sensor devices can also be used for analyzing and assaying molecules.
2. Background Art
With the advent of automated equipment for introducing compositions into wells of a microtiter plates, a number of efforts have been made to develop plates which include all of the components necessary to analyze plated cells or small molecules in a single step. For example, in Ehret, et al., Biosensors and Bioelectronics, 12:29-41 (1997), the authors described an electronic impedance-based device for measurement of cells in a liquid analyte sample. The presence of targeted cells in the same is indicated by a change in impedance between evenly sized and spaced electrode pairs to which the cells adhere. U.S. Pat. No. 6,376,233, describe how such a device in combination with other sensors would be produced in a microtiter plate format using semiconductor material as a the “plate” to provide the substrate for the measurement electrodes. Electrical conduits extend from electrodes in the '233 Patent device in various planes, and in several directions, through the semiconductor substrate.
Others have explored in using electronic methods for analyzing and assaying biological molecules and cells. For example, U.S. Pat. No. 3,890,201 describes a multichamber module-cap combination device in which electrically conductive strips in the bottom of the chambers are used for measuring the impedance of a sample of nutrient media in which aerobic microorganisms are grown, and U.S. Pat. No. 4,072,578 describes a multi-chambered module attached to an electrically non-conductive base within which electrically conductive leads completely embedded and lying flat, terminal portions at one end of the conductive leads emerging in pairs into the chamber in spaced relationship to each other to form electrodes for culturing samples of microorganisms while monitoring the impedance of the growth media.
U.S. Pat. No. 5,187,096 discloses a cell substrate electrical impedance sensor with multiple electrode arrays. Each electrode pair within the impedance sensor for measuring the cell-substrate impedance comprises one small electrode (a measuring electrode) and one large electrode (a reference electrode) on two different layers. The difference between the electrode sizes ensures that the measured impedance change relative to the impedance when no cells are present on the electrodes is directly correlated with the cell numbers and sizes, generally 20-50 cells, or even single cells attached to or grown on the measuring electrodes. Some applications of the cell sensor include the monitoring of conditions within bioreactors, within cell cultures, the testing of compounds for cytotoxicity, research of cell biology to detect cell motility, metabolic activity, cell attachment and spreading, etc. However, this impedance sensor with two layered structures is somewhat complicated with the measuring electrodes on one layer and the reference electrodes on another layer. The selected electrode area for the small electrodes limits the maximum of 50 cells being monitored.
The use of a large (reference) electrode and a small (measurement or active) electrode for cell-electrode impedance measurement was reported in many publications, including, Giaever I. and Keese C. R., “Monitoring fibroblast behavior in tissue culture with an applied electric field”, Proc. Natl. Acad. Sci. (USA), 1984, vol. 81, pp 3761-3764; Giaever I. and Keese C. R., “Micromotion of mammalian cells measured electrically”, Proc. Natl. Acad. Sci. (USA), 1991, vol. 88, pp 7896-7900; Tiruppathi C. et al, “Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function”, Proc. Natl. Acad. Sci. (USA), 1992, vol. 89, pp 7919-7923; Lo C. M. et al., “Monitoring motion of confluent cells in tissue culture”, Experimental cell research, 1993, vol. 204, pp 102-109; Lo C. M. et al, “Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing”, Biophys. J., 1995, vol. 69, pp. 2800-2807; Lo C. M. et al, “pH change in pulsed CO2 incubators cause periodic changes in cell morphology”, Experimental cell research, 1994, vol. 213, pp. 391-397; Mitra P. et al., “Electric measurements can be used to monitor the attachment and spreading of cells in tissue culture”, BioTechniques, 1991, vol. 1, pp. 504-510; Kowolenko M. et al, “Measurement of macrophage adherence and spreading with weak electric fields”, J. Immunological Methods, (1990) vol. 127, pp. 71-77; Luong J. H. et al, “Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor”, Anal. Chem.; 2001; vol: 73, pp1844-1848. For example, in the first article of cell-electrode impedance measurement (by Giaever I. and Keese C. R., “Monitoring fibroblast behavior in tissue culture with an applied electric field”, Proc. Natl. Acad. Sci. (USA), 1984, vol. 81, pp 3761-3764), the large electrode had an area ˜2 cm2 and the small electrode had an area of 3×10−4 cm2.
PCT application US01/46295 (WO 02/42766) and U.S. patent application Publication 2002/0086280 describe a similar system adapted for monitoring cell movement. At least one sensing electrode (measurement electrode) and a counter electrode are situated in a well into which a biocompatible chemical gradient stabilizing medium is introduced and into which migratory cells are placed. A migrating cell's arrival at the sensing electrode is detected by a change in impedance due to contact between the cell and a sensing electrode, which is smaller than the counter electrode. The system can be used to determine the stimulatory or inhibitory effect of test compounds on cell migration by comparing the time of arrival of a migratory cell at a sensing electrode (detected by the impedance change) in the presence of a test compound with the time of arrival of a migratory cell at a sensing electrode in the absence of a test compound.
U.S. Pat. Nos. 5,981,268 and 6,051,422 disclose a similar hybrid sensor for measurement of single cells. In this case, an array of measuring electrodes shares a common reference electrode. In order to measure single cell responses, the diameter of measuring electrode is smaller than that of a cell. The sensors can be applied to detect and monitor changes in cells as a result of cell responses to environmental and chemical challenges. However, this impedance sensor can monitor responses of only single cells. Furthermore, the sensitivity of such devices critically depends on the cell location relative to the electrodes.
U.S. patent applications 2002/0150886 and 2002/0076690 disclose the use of antibodies immobilized on interdigitated electrodes for the detection of pathogens. The interdigitated electrodes are incorporated onto a surface of a fluidic channel through which a fluid sample is passed, and binding of a pathogen to the antibody-coated electrodes can be detected by an increase in impedance between spaced electrodes.
The use of interdigitated electrodes fabricated on silicon or sapphire or glass substrates as impedance sensors to monitor cell attachment is described in papers by Ehret et al. (Biosensors and Bioelectronics 12: 29-41 (1996); Med. Biol. Eng. Computer. 36: 365-370 (1998)), Wolf et al. (Biosensors and Bioelectronics 13: 501-509 (1998)), and Henning et al. Anticancer Drugs 12: 21-32 (2001)). These methods use expensive substrates such as silicon and sapphire and, due to the electrode configurations (both electrode widths and gaps are about 50 microns), have a less than optimal efficiency, as only an average of about 50% of the cells are able to contribute to the impedance signal.
U.S. Pat. No. 6,280,586 discloses a device for measuring the presence of a component of an analyte having at least one reference sensor and at least one electrical sensor each having a measurement output connectable to an evaluation device. The reference sensor interdigitated capacitor and a reference electrode each having an electrical measurement structure are located on a common substrate. The measurement structure of the electrical sensor is connected to at least one function-specific plant or animal receptor cell serving as a biological sensor, wherein each electrical sensor measures the analyte under investigation by measuring a morphologic or physiologic property of the receptor cells. A structured, biocompatible micro porous interlayer is provided between the receptor cell and the measurement structure. The receptor is at least partially adhered to the microporous interlayer. The measurement structure of the reference sensor is free of connections of function specific receptor cells. The change of the measured property is indicative of the presence of the compound in the analyte.
U.S. Pat. No. 5,810,725 discloses a planar electrode array for stimulation and recording of nerve cells and the individual electrode impedance is in a range between 1 ohm and 100 k-ohm at a frequency of 1 kHz with an electrolytic solution comprising 1.4% NaCl. U.S. Pat. No. 6,132,683 discloses an electrode array comprising a plurality of measuring electrodes and reference electrodes for monitoring and measuring electrical potential in a neural cell sample, wherein the impedance of the reference electrode is smaller than that of measuring electrodes. However, these electrodes are not optimized for a quantitative measurement of impedance at the interface between a cell and a microelectrode.
In another type of application, direct current (DC) electrical field is used to electronically size particles, in particular, biological cells by using the well-known “coulter” counting principle. In this case, a DC current is applied to a micron or multiple-micro-size aperture. Electrical voltage change is monitored when a cell or other particle is forced through the aperture. Despite its success of the coulter principle, the device is limited in its sensitivity as well as its dynamic range in counting and sizing biological cells. See U.S. Pat. Nos. 2,656,508 and 3,259,842, and Larsen et al., “Somatic Cell Counting with Silicon Apertures”, Micro Tatal Analysis Systems, 2000, 103-106, edited by A. Van den Berg et al., 2000 Kluwer Academic Publishers:
U.S. Pat. No. 6,169,394 discloses a micro-electric detector having conductivity or impedance based measurements of a test sample placed in a microchannel. The detector includes a pair of electrodes disposed on opposing sidewalls of the microchannel to create a detection zone in the microchannel between and adjacent to the electrodes. Similarly, Song at al. demonstrated use of such microelectrodes for detecting cellular DNA content by measuring capacitance change when a cell is caused to pass by two opposing electrodes disposed on the two side walls of a microfluidic channel. See Song at al., Proc. Natl. Acad. Sci. U.S.A., 97(20):10687-90 (2000).
U.S. Pat. Nos. 5,643,742, 6,235,520 and 6,472,144 disclose systems for electrically monitoring and recording cell cultures, and for high throughput screening. The systems comprise multiple wells into each of which cells are introduced and into each of which a pair of electrodes are placed. The systems can measure the electrical conductance within each well by applying a low-voltage, AC signal across a pair of electrodes placed in the well and measuring the conductance across the electrodes, to monitor the level of growth or metabolic activity of cells contained in each well.
Others have taken different approaches to the use of impedance measurements to assay molecules in a sample. For example, Ong, et al., Sensors, 2:219-232 (2000), uses impedence changes in a circuit to detect the presence of bacteria in food. In German published application DE 39 15 290 and PCT Application WO 96/01836 devices are disclosed as having electrodes disposed on a substrate for use in detection of small molecules, especially polynucleotides. However, these devices are limited to use in specific applications, and are not intended for general laboratory research.
Other bioelectrical sensors rely on changes in capacitance or other signals as indicia of assay results. For example, U.S. Pat. No. 6,232,062 discloses a method for detecting the presence of a target sequence in a nucleic acid sample. The method comprises applying a first input signal comprising an AC component and a non-zero DC component to a hybridization complex, said hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid, said hybridization complex being covalently attached to a first electron transfer moiety comprising an electrode and a second electron transfer moiety, and detecting the presence of said target sequence by detecting the presence of said hybridization complex. Examples of the second electron transfer moieties include transition metal complexes, organic electron transfer moieties, metallocenes.
In another example, Patolsky et al, Nature Biotechnology, 19, 253-257, (2001), described a method for detection of single base mutation in DNA. With electrochemical redox labels, they measured Faradic impedance spectra for an electrode on which a primer thiolated oligonucleotide was assembled and hybridization of target DNA molecules occurred. The technique achieved a sensitivity of 10−14 mol/ml for sample DNA tested.
Bioelectrical sensors have also been adapted to use in detection of cell migration. For example, in the device of Cramer (U.S. Pat. No. 4,686,190), the passage of cells through a membrane can be detected by a sensor. However, the usefulness of the Cramer device is limited by several design limitations including, in one embodiment, the concealment of the active surface of the sensor by the membrane.
This invention aims to expand the usage and application of electrical field impedance measurement and other electronic methods for measuring and analyzing cells and molecules, non-cell particles, and biological, physiological, and pathological conditions of cells, and provides devices, apparatuses and systems for these analyses.